Refolding in the modern biopharmaceutical industry.

Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be further purified to obtain pure and active biologicals. This work focusses on refolding as a significant process step during biopharmaceutical manufacturing with an industrial perspective. A theoretical and historical background on protein refolding gives the reader a starting point for further insights into industrial process development. Quality requirements on IBs as starting material for refolding are discussed and further economic and ecological aspects are considered with regards to buffer systems and refolding conditions. A process development roadmap shows the development of a refolding process starting from first exploratory screening rounds to scale-up and implementation in manufacturing plant. Different aspects, with a direct influence on yield, such as the selection of chemicals including pH, ionic strength, additives, etc., and other often neglected aspects, important during scale-up, such as mixing, and gas-fluid interaction, are highlighted with the use of a quality by design (QbD) approach. The benefits of simulation sciences (process simulation and computer fluid dynamics) and process analytical technology (PAT) for seamless process development are emphasized. The work concludes with an outlook on future applications of refolding and highlights open research inquiries.